THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Patent metadata

Field	Value
Publication number	US-2021274726-A1
Application number	US-202117327620-A
Country	US
Kind code	A1
Filing date	May 21, 2021
Priority date	Jul 9, 2013
Publication date	Sep 9, 2021
Grant date	—

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells for treating or preventing genetic blood disorders.

First claim

What is claimed is:

1 . A method for treating or preventing a disorder associated with expression of a SCD-associated polynucleotide sequence in a subject, the method comprising (a) altering a target SCD-associated polynucleotide sequence in a cell ex vivo by contacting the SCD-associated polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target SCD-associated polynucleotide sequence, wherein the target SCD-associated polynucleotide sequence is cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the SCD-associated polynucleotide sequence.

2 . A method for treating or preventing a disorder associated with expression of a SCD-associated polynucleotide sequence in a subject, the method comprising (a) altering a target SCD-associated polynucleotide sequence in a cell ex vivo by contacting the SCD-associated polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target SCD-associated polynucleotide sequence, wherein the target SCD-associated polynucleotide sequence is cleaved; (b) contacting the cleaved target SCD-associated polynucleotide sequences with an exogenously introduced DNA repair template to initiate homology-directed repair of the target SCD-associated polynucleotide; and (c) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the SCD-associated polynucleotide sequence.

3 . A method for treating or preventing a disorder associated with expression of an HBB gene sequence in a subject, the method comprising (a) altering a target HBB gene sequence in a cell ex vivo by contacting the HBB gene sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target HBB gene sequence, wherein the target HBB gene sequence is cleaved, and (b) introducing the cell into the subject, thereby treating or preventing a disorder associated with expression of the HBB gene sequence.

4 . A method according to any one of claims 1 - 3 , wherein the disease is sickle cell disease.

5 . A method according to any one of claims 1 - 3 , wherein the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

6 . A method according to claim 4 , wherein the Cas protein is Streptococcus pyogenes Cas9 protein.

7 . A method according to any one of claims 1 - 3 , wherein the Cas protein is complexed with the one to two ribonucleic acids.

8 . A method according to any one of claims 1 - 3 , wherein the target motif is G(N) 19 NGG or (N) 20 NGG.

9 . A method according to any one of claims 1 - 3 , wherein the cell is selected from the group consisting of a peripheral blood cell, a stem cell, a pluripotent cell, a hematopoietic stem cell, a CD34+ cell, a CD34+ mobilized peripheral blood cell, a CD34+ cord blood cell, a CD34+ bone marrow cell, a CD34 + CD38-Lineage-CD90 + CD45RA − cell, a primary human cell, a non-transformed human cell, and combinations thereof.

10 . A method according to any one of claims 1 - 3 , wherein the Cas protein is encoded by a nucleic acid sequence comprising a modified nucleic acid selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate.

11 . A method according to any one of claims 1 - 3 , wherein at least one of the ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate.

12 . A method according to any one of claims 1 - 3 , wherein the target motif is located between position 5246806 and position 5248263 of human chromosome 11.

Assignees

• Harvard College
• Childrens Medical Ct Corp

Inventors

• Musunuru Kiran
• Cowan Chad A
• Rossi Derrick J

Classifications

• B05B7/0075

  Nozzle arrangements in gas streams · CPC title

• E01H1/101

  Hydraulic loosening or dislodging, combined or not with mechanical loosening or dislodging, e.g. road washing machines with brushes or wipers (E01H1/005 takes precedence; cleaning rails E01H8/105, E01H8/125; applying liquids in general E01H3/00; scrubbing machines for floors or similar surfaces A47L11/26 - A47L11/30) · CPC title

• E01H2001/0881

  Details for pneumatically dislodging or removing not related to the mouth-piece · CPC title

• B05B1/005

  Nozzles or other outlets specially adapted for discharging one or more gases · CPC title

• A47L11/4088

  Supply pumps; Spraying devices; Supply conduits · CPC title